The c-sis gene encodes a precursor of the B chain of platelet-derived growth factor.

The relationship between platelet-derived growth factor (PDGF) and the proto-oncogene c-sis has been determined by amino acid sequence analysis of PDGF and nucleotide sequence analysis of c-sis genomic clones. The nucleotide sequences of five regions of the human c-sis gene which are homologous to sequences of the transforming region (v-sis) of simian sarcoma virus (SSV) were determined. By alignment of the c-sis and v-sis nucleotide sequences the predicted amino acid sequence of a polypeptide homologous to the putative transforming protein p28sis of SSV was deduced. Both predicted sequences use the same termination codon and additional coding sequences may lie 5' to the homologous regions. Amino acid sequence analysis of the PDGF B chain shows identity to the amino acid sequence predicted from the c-sis sequences over 109 amino acid residues. Polymorphism may exist at two amino acid residues. These results suggest that c-sis encodes a polypeptide precursor of the B chain. A partial amino acid sequence of the PDGF A chain is also described. This chain is 60% homologous to the B chain and cannot be encoded by that part of c-sis which has been sequenced but could be encoded by sequences which lie 5' to the five regions of v-sis homology in c-sis, or at a separate locus.     

Nucleotide sequence of transforming human c-sis cDNA clones with homology to platelet-derived growth factor.

Three c-sis cDNA clones were obtained from polyadenylated RNA of a human T-cell lymphotropic virus (HTLV) type I transformed cell line. Two clones, designated pSM-1 and pSM-2, have cDNA inserts of 2498 and 2509 base pairs (bp), respectively, excluding the sizes of the guanylate tails, and the polyadenylate tracts. These clones are shorter than the estimated size of the c-sis mRNA of 4200 bp. Both of these clones can transform NIH 3T3 cells. The third clone, designated pSM-3 has a cDNA insert of 1421 bp and lacks transforming activity. The sequence of clone pSM-1 reveals a single long open reading frame (nucleotides 118-840) encoding chain A of platelet-derived growth factor, and two segments with homology to v-sis (nucleotides 182-871 and 1021-1325). Sequence homology is noted in the 3' untranslated region to the corresponding regions of the beta 1 interferon (IFN), human and murine beta-nerve growth factor (NGF), human interleukin 2 (IL2) genes, and tubulin pseudogenes. However, no typical AATAAA polyadenylation signal is present. An alternating (dCdA)n X (dGdT)n sequence is present in the 3' flanking cellular sequences similar to those in the corresponding position of the human proenkephalin gene, in the first intron of the gamma-IFN gene, and the second intron of the beta-NGF gene.     

Agents that increase cAMP accumulation block endothelial c-sis induction by thrombin and transforming growth factor-beta

Endothelial cells express the product of the c-sis gene, which encodes the B-chain of platelet-derived growth factor (PDGF). Through local production of growth factors such as PDGF in vascular sites, endothelial cells may stimulate proliferation of adjacent cells through a paracrine mechanism. Previously, we have shown that the expression of c-sis mRNA and release of growth factor activity by human renal endothelial cells is induced by thrombin. We now show that another agent of possible importance in mediating proliferation of cells adjacent to the endothelial cell layer, transforming growth factor-beta (TGF-beta), also induced c-sis expression in these cells. In addition, we have studied the effect of agents that increase intracellular cAMP levels upon the induction of endothelial cell c-sis mRNA. The adrenergic agonists isoproterenol and norepinephrine blocked the elevation of cellular c-sis mRNA accompanying exposure to either thrombin or TGF-beta. This effect was mediated through beta-adrenergic receptors, since propranolol but not phentolamine reversed the inhibition. Forskolin, a direct activator of adenylate cyclase, also blocked induction of c-sis mRNA by thrombin and TGF-beta and inhibited the release of PDGF activity into the media of these cells. Basal, as well as stimulated c-sis mRNA levels were attenuated by these agents that increase cellular cAMP levels. These data suggest that increased cAMP production inhibits the expression of c-sis encoded mitogens by endothelial cells, and that c-sis expression is subject to bidirectional regulation in these cells.     

Modification of proto-oncogene expression by phorbol esters in human dermal microvascular endothelial cells.

Following activation with the inflammatory mediator phorbol myristate acetate (PMA), human microvascular endothelial cells (DMEC) is olated from the human dermis (DMEC) rapidly and dramatically convert from a classical epithelioid morphology to a spindle-shaped configuration. This is accompanied by changes in the organization of gap junctions and the vimentin and actin cytoskeletons. This report describes the sequential changes in the expression of four proto-oncogenes, c-fos, c-myc, c-sis and H-ras in DMEC following PMA exposure. The synthesis of c-fos mRNA was transiently induced by PMA from a basal concentration below the limit of detection to a maximum at 60 min., declining to the unstimulated level within 2 hrs. Synthesis of c-myc mRNA declined continuously and reached 37% of control levels over 16 hrs. Expression of c-sis which encodes for the B chain of platelet-derived growth factor, also declined to 34% of the control value over 16 hrs. There was no change in the synthesis of H-ras mRNA nor of beta-actin mRNA which was used as a control. The expression of c-myc in normal DMEC was compared to a human dermal microvascular cell line transformed by SV-40 (TREND). The TREND cell line maintains a permanent spindle-shaped configuration under all growth conditions and multiplies faster than DMEC. In contrast to the non-transformed cell cultures, expression of c-myc in TREND cells was induced by PMA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Partially transformed, anchorage-independent human diploid fibroblasts result from overexpression of the c-sis oncogene: mitogenic activity of an apparent monomeric platelet-derived growth factor 2 species.

A human c-sis cDNA in an expression vector was introduced into human diploid fibroblasts by transfection or electroporation. Fibroblast clones showing an aberrant, densely packed colony morphology were isolated and found to overexpress a 3.6-kilobase sis mRNA species and associated immunoprecipitable platelet-derived growth factor (PDGF) 2 proteins. Parallel analyses in cell clones of sis mRNA expression and colony formation in agar indicated that, above a threshold, a linear, positive correlation existed between sis overexpression and acquired anchorage independence. The sis-overexpressing cells formed transient, regressing tumor nodules when injected into nude mice, consistent with the finite life span which they retained. Protein products generated from the transfected c-sis construct in two overexpressing clones were immunoprecipitated with anti-human PDGF antibodies. One clone contained an apparent PDGF dimer of 21 kilodaltons; the second clone contained only an apparent PDGF monomer of 12 kilodaltons, which was shown to account for all of the mitogenic activity present in the cells, essentially all of which was concentrated in the membrane fraction. The results demonstrate a clear link between sis overexpression and acquisition of a partially transformed, anchorage-independent phenotype, and when combined with previous observations of sis overexpression in human tumors, clearly implicate sis overexpression as a genetic mechanism which contributes to human cell transformation.     

Structure of the murine c-sis proto-oncogene (Sis, PDGFB) encoding the B chain of platelet-derived growth factor

The murine proto-oncogene c-sis (Sis, PDGFB), encoding the B chain of platelet-derived growth factor, has been cloned. Its structure, with seven exons spanning approximately 20 kb, closely resembles that of the human and feline homologs. The predicted amino acid sequence of murine PDGF-B has residues 89% identical to those of human PDGF-B. A noncoding region at the start of exon 7, which is deleted by alternative splicing during the generation of the viral v-sis oncogene, is highly conserved in human, mouse, and cat and may represent an important regulatory element.     

The cell cycle dependence of c-sis gene expression: artifactual conclusions in cells prepared by chemical but not physical techniques

The c-sis oncogene encoding the B-chain of platelet-derived growth factor (PDGF) may be involved in an autocrine growth stimulation of tumours expressing the PDGF receptor, such as glioblastomas and sarcomas. To investigate whether expression of c-sis RNA is regulated in a cell cycle dependent manner, human A172 glioblastoma cells were synchronized by either centrifugal elutriation or chemical blockage with the DNA synthesis inhibitors hydroxyurea or aphidicolin. In non-perturbed elutriated cells, c-sis RNA levels were lower in the S phase of the cell cycle than in the G1 phase. In contrast, the chemically synchronized cells revealed a transient rise in c-sis RNA shortly after drug release, in early S phase. The RNA changes occurring after release from drug inhibition represent cell recovery from drug induced metabolic disturbances rather than true cell cycle dependent effects.